Structure-activity relationship study of host-specific phytotoxins (AM-toxin analogs) using a new assay method with leaves from apple meristem culture

AM-toxins are host-specific phytotoxins of the Alternaria alternata apple pathotype, which induce necrosis on apple leaves. In this study, we developed a new assay to measure the necrotic activity of AM-toxin analogs using cultured leaves from meristem cells. This method was not only more sensitive to AM-toxin I, but also more reliable than the previous one that used tree leaves due to the homogeneous nature of cultured leaves and to the method of application of toxins. Using this assay method we investigated a structure-activity relationship of AM-toxin analogs synthesized in this study. Most residues and the macrocyclic ring structure were strictly recognized by AM-toxin putative receptor, whereas the L-Ala binding subsite of the receptor allowed for side chain structures with various stereoelectronic properties. These findings are important for designing ligands for further experimental probing of the nature of the receptor.     

Cloning and characterization of a cyclic peptide synthetase gene from Alternaria alternata apple pathotype whose product is involved in AM-toxin synthesis and pathogenicity

Afternaria afternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide host-specific toxin, AM-toxin. PCR (polymerase chain reaction), with primers designed to conserved domains of peptide synthetase genes, amplified several products from A. alternata apple pathotype that showed high similarity to other fungal peptide synthetases and were specific to the apple pathotype. Screening of a Lambda Zap genomic library with these PCR-generated probes identified overlapping clones containing a complete cyclic peptide synthetase gene of 13.1 kb in length with no introns. Disruption of this gene, designated AM-toxin synthetase (AMT), by transformation of wild-type A. afternata apple pathotype with disruption vectors resulted in toxin-minus mutants, which were also unable to cause disease symptoms on susceptible apple cultivars. AM-toxin synthetase is therefore a primary determinant of virulence and specificity in the A. alternata apple pathotype/apple interaction.     

Expression profiles of genes encoded by the supernumerary chromosome controlling AM-toxin biosynthesis and pathogenicity in the apple pathotype of Alternaria alternata

The apple pathotype of Alternaria alternata produces host-specific AM-toxin and causes Alternaria blotch of apple. Previously, we cloned two genes, AMT1 and AMT2, required for AM-toxin biosynthesis and found that these genes are encoded by small, supernumerary chromosomes of <1.8 Mb in the apple pathotype strains. Here, we performed expressed sequence tag analysis of the 1.4-Mb chromosome encoding AMT genes in strain IFO8984. A cDNA library was constructed using RNA from AM-toxin-producing cultures. A total of 40,980 clones were screened with the 1.4-Mb chromosome probe, and 196 clones encoded by the chromosome were isolated. Sequence analyses of these clones identified 80 unigenes, including AMT1 and AMT2, and revealed that the functions of 43 (54%) genes are unknown. The expression levels of the 80 genes in AM-toxin-producing and nonproducing cultures were analyzed by real-time quantitative polymerase chain reaction (PCR). Most of the genes were found to be expressed in both cultures at markedly lower levels than the translation elongation factor 1-alpha gene used as an internal control. Comparison of the expression levels of these genes between two cultures showed that 21 genes, including AMT1 and AMT2, were upregulated (>10-fold) in AM-toxin-producing cultures. Two of the upregulated genes were newly identified to be involved in AM-toxin biosynthesis by the gene disruption experiments and were named AMT3 and AMT4. Thus, the genes upregulated in AM-toxin-producing cultures contain ideal candidates for novel AM-toxin biosynthetic genes.     

Cyclic peptides. II. Synthesis of AM-Toxin I

To confirm the structure of AM-toxin I (a phytotoxic cyclotetradepsipeptide) the proposed peptide was prepared by a conventional method. The synthetic peptide and natural AM-toxin I were identical as regards t.l.c., u.v., mass spectra and biological activity in causing necrosis on apple leaves. A prepared dimer of AM-toxin I showed extremely weak activity; the relationship between the ring size and biological activity of AM-toxin I is discussed.     

Selection of mutants resistant to Alternaria blotch from in vitro -cultured apple shoots irradiated with X- and γ-rays

We produced mutants resistant to Alternaria blotch disease in several cultivars of apple (Malus × domestica Borkh.) by irradiation with X- or γ-rays. An efficient in vitro assay method was established using chemically-synthesized AM-toxin I of Alternaria alternata (Fr.) Keissler to screen for mutants resistant to Alternaria blotch disease. The frequency of necrotic lesions was investigated by applying various concentrations of AM-toxin I to leaf discs of the first, third, and fifth leaves from the shoot apex of several apple cultivars, including Jonathan, Fuji, Oorin, and Indo. In vitro-grown apple shoots of susceptible cultivars were then treated with various doses of X- or γ-ray irradiation. Several mutants resistant to AM-toxin I were obtained by combining the techniques for tissue culture of apple shoots with the AM-toxin I screening method. Following a repeat second screening test with AM-toxin I, mutant plants were sprayed with a spore suspension of A. alternata and found resistant to be the fungal pathogen. These mutants showed normal phenotypic appearance, and so far, no difference has been observed between the original plants and mutants except for the susceptibility to Alternaria blotch.     

Nuclear magnetic resonance study of ring conformations of cyclic tetradepsipeptide AM-toxins and related peptides

Cyclic tetradepsipeptides, AM-toxin I and II, are the host-specific phytotoxins of Alternaria mali. In order to elucidate conformation-toxicity relationships, we analyzed the 270-MHz proton nmr spectra of AM-toxins and hydrogenated analogs, (D -Ala²)AM-toxin I (toxic) and (L -Ala²)AM-toxin I (not toxic), in (C²H₃)₂SO. These cyclic tetradepsipeptides do not contain N-substituted amino acid residues, and all the peptide and ester groups have been found to be transoid. Two conformers with very unequal populations have been found for AM-toxin I and II; the C^β?C^α? C?O conformations of the Dha² residues are nonplanar S-trans in the major conformer and nonplanar S-cis in the minor conformer. Only one ring conformation has been found for each of (L -Ala²) and (D -Ala²)AM-toxin I. (L -Ala²)AM-toxin I takes a C₄-type ring conformation; all the C?O groups and C^α-H bonds are oriented to the same side of the ring. (D -Ala²)AM-toxin I takes a new ring conformation; the side chain and C?O group of the L -Amp¹ residue are oriented to the same side of the ring. This new conformation is also found for the major conformers of AM-toxin I and II and thus appears to be required for the toxicity. The ring conformations of Tyr(OCH₃)¹-bearing analog tetradepsipeptides have been found to be much the same as those of Amp¹-bearing depsipeptides. Furthermore, on the basis of the two distinct conformations of (D -Ala²) and (L -Ala²)AM-toxin I, an empirical rule is proposed for the stable ring conformations of cyclic tetra-D ,L -peptides, not containing N-substituted amino acid residues.     

G protein signaling mediates developmental processes and pathogenesis of Alternaria alternata

A G protein alpha subunit gene (AGA1) has been cloned and characterized from a toxigenic and necrotrophic Alternaria alternata pathogen. Targeted disruption of AGA1 in the apple pathotype of A. alternata gave rise to mutants that differed in colony and conidial morphology as well as sporulation. The conidia of wild type and deltaAGA1 mutants showed equal germination on cellulose membranes. However, wild-type germ tubes formed readily from different points around the conidia, grew randomly, and were often branched, whereas those of the mutants formed only at one or both ends of the conidia and tended to grow in straight paths. Targeted disruption of AGA1 also resulted in reduction of pathogenicity on apple leaves, although the mutant produced host-specific AM-toxin, a fungal secondary metabolite associated with pathogenicity of the pathogen, at levels similar to the wild-type strain. Measurement of the intracellular cAMP levels of the mutant revealed that it was consistently higher than that of the wild type, indicating that AGA1 negatively regulates cAMP levels similar to mammalian Galphai systems. These results indicate that the signal transduction pathway represented by AGA1 appears to be involved in developmental pathways leading to sporulation and pathogenesis of A. alternata.     

Light stimulated activity of nitrate reductase in apple roots

The nitrate reductase activity in the roots of apples grownin the dark for 48 hr prior to assay ws lower than the grownunder normal conditions of alternating 12 hr light and 12 hrdarkness. Removal of leaves, cincturing the stem, and applicationof a photosynthetic inhibitor also lowered reductase activityin the light. (Received July 9, 1973; )     

A Host-specific, Nitrogen Fixation Mutant of Bradyrhizobium: Physiology on Three Host Plants

NC92 #284 is a transposon mutant of Bradyrhizobium sp. (Arachis)strain NC92 and has a host-specific fixation phenotype. It appearsto be ineffective on the host pigeonpea (90% reduction in shootN compared to that of the wild type), but partially effectiveon two other host plants, groundnut and siratro (50% and 20%reduction in shoot N compared to the wild type, respectively).To understand this phenomenon of host-specific fixation, thephysiological basis of the phenotypes was investigated. Host-dependentdifferences in symbiotic effectiveness were largely explainedby the degree to which nitrogenase activity was impaired inthe various #284 symbioses. Nodulation and the onset of nitrogenfixation were found to be delayed on all three hosts, but tothe greatest degree on pigeonpea. The specific activity of nitrogenaseper gram nodule was also reduced on all three hosts, again tothe greatest extent on pigeonpea. By contrast, the carbon costsand relative efficiencies of each symbiosis were similar tothe wild type. The results indicate that the host-specific fixationphenotype of #284 is reflected in a quantitative reduction inthe amount of N₂ fixed. Thus the phenotypes reflect the differentability of the three host plants to tolerate or support the#284 mutation, rather than a defect in a specific interactionbetween #284 and a particular host plant. Key words: Bradyrhizobium, nodules, nitrogenase activity, relative efficiency, Arachis hypogaea, Cajanus cajan, Macroptilium atropurpureum     

Ecology of Staphylococcus aureus: comparative characterization of strains isolated from man, cattle and sheep in Bulgaria and in the GDR

Staphylococcus aureus strains from man, cattle and sheep have been differentiated by biochemical tests and by phage typing. Strains from pathological material of human origin mostly belong to the host-specific variety hominis, strains from mastitis in cattle mostly belong to the host-specific variety bovis. However, there are also strains from cattle which cannot be allotted to one of the known host-specific varieties and also strains which belong to the host-specific variety hominis. Strains from mastitis in sheep clearly differ from strains of human and bovine origin; these strains are designated as variety ovis. The frequency distribution of the host-specific varieties in man, cattle and sheep is the same in Bulgaria and in the GDR.     

Photosynthetic bradyrhizobia from Aeschynomene spp. are specific to stem-nodulated species and form a separate 16S ribosomal DNA restriction fragment length polymorphism group.

We obtained nine bacterial isolates from root or collar nodules of the non-stem-nodulated Aeschynomene species A. elaphroxylon, A. uniflora, or A. schimperi and 69 root or stem nodule isolates from the stem-nodulated Aeschynomene species A. afraspera, A. ciliata, A. indica, A. nilotica, A. sensitiva, and A. tambacoundensis from various places in Senegal. These isolates, together with 45 previous isolates from various Aeschynomene species, were studied for host-specific nodulation within the genus Aeschynomene, also revisiting cross-inoculation groups described previously by D. Alazard (Appl. Environ. Microbiol. 50:732-734, 1985). The whole collection of Aeschynomene nodule isolates was screened for synthesis of photosynthetic pigments by spectrometry, high-pressure liquid chromatography, and thin-layer chromatography analyses. The presence of puf genes in photosynthetic Aeschynomene isolates was evidenced both by Southern hybridization with a Rhodobacter capsulatus photosynthetic gene probe and by DNA amplification with primers defined from photosynthetic genes. In addition, amplified 16S ribosomal DNA restriction analysis was performed on 45 Aeschynomene isolates, including strain BTAi1, and 19 reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii, and other Bradyrhizobium sp. strains of uncertain taxonomic positions. The 16S rRNA gene sequence of the photosynthetic strain ORS278 (LMG 12187) was determined and compared to sequences from databases. Our main conclusion is that photosynthetic Aeschynomene nodule isolates share the ability to nodulate particular stem-nodulated species and form a separate subbranch on the Bradyrhizobium rRNA lineage, distinct from B. japonicum and B. elkanii.     

干旱下接种根际促生细菌对苹果实生苗光合和生理生态特性的影响

本试验研究了接种根际促生细菌(PGPR)对干旱条件下植物光合和生理生态特性的影响,以期为PGPR在植物抗旱中的应用提供理论依据.采用盆栽试验,以苹果实生幼苗为供试植物,以经过筛选得到的既具有1-氨基环丙烷-1-羧酸(ACC)脱氨酶活性又具有较强溶磷能力的根际促生菌YX₂为供试菌株,设置正常水分(CK)、轻度干旱(LD)、中度干旱(MD)和重度干旱(SD),其相应含水量分别为田间持水量的70%～80%、55%～65%、40%～50%、25%～35%,研究不同程度干旱胁迫条件下接种YX₂对苹果实生幼苗光合和生理生态特性的影响.结果表明: 与未接种处理相比,干旱环境下接种YX₂提高了苹果幼苗叶片的相对含水量、叶绿素含量、抗氧化酶活性、叶绿素荧光值、气孔导度和光合性能,降低了相对电导率、渗透调节物质和丙二醛的积累,缓解了干旱胁迫对净光合速率的抑制,增强了抗氧化系统的防御能力,减少了细胞膜过氧化伤害,提高了植株抗旱性能.     

苹果密植园与间伐园树冠层内叶片光合潜力比较

通过对成龄苹果密植园和间伐园树冠不同层次和部位叶片光合潜力及辐射通量密度、叶片N含量和比叶重等指标的比较分析,研究了苹果园改造前后辐射能和氮素利用效率差异及其与产量品质的关系.结果表明：间伐显著改善了冠层内的辐射环境,间伐园冠层内的辐射分布明显比密植园均匀,相对辐射通量密度小于30%的无效光区接近0,而密植园冠层内的最低相对辐射通量密度为17%,在相对高度03以下均为无效光区;间伐园内冠层叶片的光合效率显著提高,间伐园树冠中、下部叶片的光合速率比密植园分别提高了78%和102%;叶片的最大羧化速率和最大电子传递速率也有较大幅度的提升.苹果园冠层叶片的光合效率与叶片N含量存在显著的相关关系,而叶片N含量又与辐射通量密度存在显著的相关关系,因此,可根据冠层叶片相对N含量的垂直分布间接和定量地判断叶片的光合效率或相对辐射通量密度的空间分布.     

Cyclic peptides. XXVI. Synthesis of AM-toxin II analogs by cyclization through ester bond formation

In order to explore the route for the preparation of cyclodepsipeptide by cyclization through an ester bond formation, two analogs of AM-toxin II, cyclotetradepsipeptide, were synthesized. As a preliminary experiment, synthesis of [L-Phe3, L-Ser(Bzl)4]-AM-toxin II, containing L-Phe and L-Ser(Bzl) in place of L-App (2-amino-5-phenyl-pentanoic acid) and delta Ala (alpha, beta-dehydroalanine), respectively, was attempted. Cyclization of H-L-Hmb-L-Phe-L-Ser(Bzl)-L-Ala-OH in CH2Cl2 at 10 mM concentration using water-soluble carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) successfully gave a cyclic monomer in 16% yield. Cyclization of H-L-Hmb-L-App-L-Ser(Bzl)-L-Ala-OH under the same conditions also afforded a cyclic monomer, [L-Ser(Bzl)4]AM-toxin II, in 19% yield. Analytical parameters of these cyclic monomers obtained were identical to those of the authentic samples obtained by cyclization through a peptide bond formation.     

In vitro sulfotransferase activity of NodH, a nodulation protein of Rhizobium meliloti required for host-specific nodulation.

Early stages of nodulation involve the exchange of signals between the bacterium and the host plant. Bacterial nodulation (nod) genes are required for Rhizobium spp. to synthesize lipooligosaccharide morphogens, termed Nod factors. The common nod genes encode enzymes that synthesize the factor core structure, which is modified by host-specific gene products. Here we show direct in vitro evidence that Rhizobium meliloti NodH, a host-specific nodulation gene, catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the terminal 6-O position of Nod factors, and we show substrate requirements for the reaction. Our results indicate that polymerization of the chitooligosaccharide backbone likely precedes sulfation and that sulfation is not absolutely dependent on the presence or the particular structure of the N-acyl modification. NodH sulfation provides a tool for the enzymatic in vitro synthesis of novel Nod factors, or putative Nod factors intermediates, with high specific activity.     

Stimulative Production of Flaviolin by Phoma wasabiae

To investigate the structure-activity relationships of host-specific HMT- and PM-toxins, 8 mimics of PM-toxin A, a component of the host-specific corn pathotoxin produced by Phyllosticta maydis, were synthesized as stereoisomeric mixtures. All the mimics, except for PM-7137, had four β-ketol groups spaced by tri- and tetra-methylenes, which is shorter than the penta-methylenes involved in native PM-toxins. A comparison of their biological activity clearly demonstrated the general structural features necessary for potent activity: four β-ketol groups were necessary with a spacing of chains equal to or longer than penta-methylene.     

Physical and kinetic properties of photosynthetic phosphoenolpyruvate carboxylase in developing apple fruit

Phosphoenolpyruvate carboxylase (PEPC) was partially purified from young developing apple fruit, cultivars Golden Delicious and Cox's Orange Pippin. Freeze-drying of tissue reduced the yield of PEPC activity compared to samples stored at 4°. Activities measured by H¹⁴CO₃⁻ incorporation exceeded the spectrophotometric assay for the enzyme with coupled NADH-malate dehydrogenase (MDH) by up to 60%. The enzyme could be stored at −16° with glycerol and bovine serum albumin for several months without loss of activity. Thermal inactivation of PEPC occurred after heating to 75° for 3 min when MDH was still slightly active. Inhibition of PEPC activity by endogenous phenolics could be prevented by grinding in liquid nitrogen in the presence of polyvinylpyrrolidine and dithiothreitol. Apparent K_m (PEP) and V_max values compared more favourably with those obtained from a C₃-species (spinach) than from a C₄-species (maize). l-Malate (5 mM) inhibited fruit PEPC by 22%; this was decreased to 12% by addition of glucose-6-phosphate (2 mM). From kinetic and effector experiments PEPC in the apple fruit is concluded to be a non-C₄ photosynthetic enzyme.     

Changes in photosynthetic characteristics during leaf development in apple

A comprehensive developmental survey of leaf area, chlorophyll, photosynthetic rate, leaf resistance, transpiration ratio, CO₂ compensation point and photorespiration was conducted in apple. The largest changes in each of the photosynthetic characteristics studied took place during the earliest stages of leaf development, coinciding with the period of greatest leaf expansion and chlorophyll synthesis. During early development, photosynthesis increased 5-fold, reaching a maximum rate of 40 mg CO₂ dm^-2 hr^-1 at a leaf plastochron index (LPI) of 10. During this same period, leaf resistance, transpiration ratio, CO₂ compensation point and mesophyll resistance decreased, while carboxylation efficiency increased. Two especially interesting aspects of the data discussed are simultaneous changes that occur at a LPI of 10 and 12 in all of the photosynthetic characteristics examined and an apparent decrease in photorespiration as leaves age. From our results it is clear that stage of leaf development is an important factor affecting the rate of photosynthesis and photorespiration.Scientific Paper No. 5687, College of Agriculture, Washington State University, Pullman. This work is supported by the National Science Foundation Grant 80-10958 and the Columbia River Orchards Foundation.     

Photosynthetic patterns of Cetraria cucullata (Bell.) Ach. at Anaktuvuk Pass,Alaska

Summary The daily photosynthetic patterns of Cetraria cucullata were followed over the 1976 summer period at Anaktuvuk Pass, Alaska. With the exception of rainy peroids, the lichen exhibited a strong diurnal pattern with peak photosynthetic activity occurring between 0300 and 0700 h. This correllated with periods of maximal lichen water retention and the presence of direct solar radiation. When the lichen was moist, a strong gradient in photosynthetic activity was observed with no activity in the lichen bases and maximal activity in the lichen tips.